This proposal consists of three interrelated objectives designed to examine critical steps in the early embryonic development and differentiation of hematopoietic cells in the leopard frog. The first objective is to use transplantation of chromosomally labeled tissue anlagen to define the precise anatomical origin of pluripotential hematopoietic stem cells. Following localization, the parameters leading to initial stem cell restriction and determination will be investigated. The second objective is to examine the primary interactions between stem cells and specific reticulo-endothelial microenvironments leading to the commitment of stem cells to particular lines of differentiation. This objective will be accomplished by in vitro combinations of stem cells with thymus epithelium or pronephric, mesonephric and liver parenchyma. Ultrastructural studies will be used to assess direct cell to cell interactions. The third objective is to examine regulatory interactions between thymus and differentiated hematopoietic cell lines. Early thymectomy will be used to characterize hematopoietic ontogeny in the absence of the thymus. Together the experiments will analyze three steps in hematopoietic ontogeny - the initial differentiation of stem cells, the commitment of stem cells to particular lines of development and the regulation of these differentiated cells.